West Nile Virus (WNV) non-structural protein 3 (NS3) is a multifunctional enzyme critical for viral replication and represents a highly well-conserved antiviral target. NS3 consists of an N-terminal serine protease (NS3pro), which relies on the NS2B cofactor for polyprotein processing, and a C-terminal superfamily 2 helicase (NS3hel) responsible for ATP-dependent RNA unwinding and associated NTPase/RTPase activities. Increasing evidence shows functional crosstalk between these domains, supporting the dual target inhibition as a promising antiviral approach. This study describes a structure-based virtual screening (SBVS) campaign using an in-house library (EMAC-DB) targeting both NS3hel and NS3pro. Following an integrated CADD workflow, we identified a new series of 4,5-dihydropyrazole derivatives as dual-site NS3 inhibitors. In vitro experiments confirmed that these compounds inhibit both NS3hel and NS3pro enzymatic activities at low micromolar concentrations. Notably, compound 5e emerged as a key candidate, demonstrating the highest potency and significantly reducing WNV replication in HuH-7 cells, with an EC50 of 0.42 ± 0.1 μM. These findings suggest that two mechanisms contribute to viral replication inhibition. Further investigations revealed that NS2B-NS3pro inhibition occurred through a non-competitive mechanism, whereas NS3hel inhibition was non-competitive with respect to ATP and competitive with respect to dsDNA, supporting the hypothesis that the compound interacts with the RNA-binding cleft of NS3. Overall, this work provides a structural and mechanistic framework for the optimization of 4,5-dihydropyrazole-based dual NS3 inhibitors, highlighting the importance of a dual-acting inhibitory strategy to potentially reduce the development of resistance.

A small library of differently substituted chromones was successfully synthesized and structurally characterized. All compounds were evaluated for their inhibitory potency and selectivity toward human cancer-associated carbonic anhydrase isoforms IX and XII, as well as the off-target isoforms I and II. Compounds 4a, 4g, 4j, and 4k selectively inhibited cancer-associated isoforms IX and XII, with no activity against the off-target isozymes I and II. Among them, compound 4k was the most potent and isozyme-selective inhibitor, with Ki 0.31 µM for hCA IX and 0.24 µM for hCA XII. To estimate drug-likeness, in silico ADMET predictions were performed, indicating that all compounds possess physicochemical and pharmacokinetic properties within the acceptable ranges. Molecular docking studies on the hCA IX isoform highlighted an optimal orientation within the binding pocket, with the chromene moiety positioned toward the zinc ion. In cellular assays 4a, 4g, 4j, and 4k selectively inhibited metabolic activity in HepG2 cells expressing hCA IX in normal conditions, whereas no activity was observed in Caco-2 cells lacking hCA IX expression.

Inflammatory diseases impose a significant global healthcare burden, necessitating the development of effective anti-inflammatory agents. Many clinically used drugs are isolated from plants or derived from natural products via semi-synthetic procedures. Among natural products, coumarins have demonstrated broad-spectrum pharmacological activities. This study investigates the anti-inflammatory mechanisms of an uncommon natural coumarin, 7-methoxy-8-isopentenyloxycoumarin (7MI), isolated from the seed of the higher Mediterranean plant Magydaris pastinacea. 7MI's effects were assessed in LPS-stimulated Raw264.7 macrophages using immunoblotting, RT-qPCR, ChIP assay, chase assay, reporter assay, ELISA, phosphatase activity assay, and transfection. 7MI significantly reduced the release of pro-inflammatory cytokines, including IL-6 and MCP-1, and suppressed cyclooxygenase-2 (COX-2) expression. Mechanistically, 7MI attenuated canonical LPS-induced TLR4 signaling by inhibiting the parallel activation of the NF-κB and MAPK/AP-1 pathways. 7MI blocked NF-κB p65 nuclear translocation and its transcriptional activity. Concurrently, 7MI enhanced MAPK phosphatase-1 (MKP-1) activity, which deactivated p38MAPK, thereby dampening activation of AP-1 and the synergistic transcription factor CCAAT/enhancer-binding protein (C/EBP)β. Beyond transcriptional suppression, 7MI exerted a distinct post-translational effect by accelerating COX-2 proteasomal degradation through inhibition of the deubiquitinases USP7 and USP22, leading to enhanced K48-linked polyubiquitination of the COX-2 protein. Crucially, in vivo administration of 7MI dramatically improved survival rate and mitigated multi-organ damage in a murine model of fatal sepsis. These findings suggest that 7MI exerts broad-spectrum anti-inflammatory effects by simultaneously suppressing canonical TLR4-driven transcriptional cascades to halt de novo inflammatory mediator synthesis and by promoting pro-inflammatory protein clearance, highlighting its potential as a lead compound for LPS-associated inflammatory disorders.

Starting from the crystal structure of a Zika virus (ZIKV) NS2B-NS3 protease with bound allosteric inhibitor, we generated dynamic pharmacophore models derived from representative molecular dynamics (MD) conformations to capture key interaction patterns. These models guided a pharmacophore-based virtual screening of commercial libraries, including commercial compounds and approved drugs, enabling the rapid selection of virtual hit compounds. Biochemical evaluation identified several inhibitors, with the antiretroviral drug Nelfinavir emerging as the most promising compound. Nelfinavir displayed the strongest inhibition of ZIKV and West Nile virus (WNV) proteases and showed antiviral activity in cell-based assays, with EC50 values of 1.79 ± 0.01 µM on ZIKV and 3.01 ± 1.25 µM in WNV, supporting its relevance as a repositionable scaffold for modulation of ZIKV and WNV infections. The activity observed across flavivirus proteases highlights the value of targeting conserved allosteric regions. Overall, this integrated computer aided drug design (CADD) strategy demonstrates the power of dynamic, structure-based pharmacophores to uncover novel allosteric inhibitors and accelerate antiviral drug repurposing.

Nucleoside triphosphate analogues in which the γ-phosphate has two different non-cleavable lipophilic alkyl residues were observed to potently inhibit HIV-1 replication in vitro. We report that a series of γ-monoalkyl triphosphate analogues of acyclic nucleoside phosphonates inhibits the RNA-dependent RNA polymerase (RdRp) function of the SARS-CoV-2 nsp12 protein. These nucleotide analogues were synthesized with multiple nucleobase derivatives, sharing one alkyl group of two different lengths on the γ-phosphonate. The enzymatic PAGE-based assay of SARS-CoV-2 nsp12 with cofactors nsp7/8 revealed IC50 values in the low micromolar range, whose potency depended on the length of the alkyl group, with no incorporation of the analogues and complete loss of enzymatic function. Competition assay revealed that the tested nucleotide analogues do not compete with the natural triphosphate nucleotides independently of sequence complementarity, hence acting as non-nucleoside inhibitors. By multiple techniques, we demonstrated that the inhibitors induce the dissociation of the nsp7 cofactor from the nsp12 subunit. Molecular docking and molecular dynamic simulations supported this mechanism identifying a putative binding site in an interface cavity between nsp12-nsp8 and nsp7, where the compound forms both hydrogen bonds and hydrophobic interactions. Different prodrugs of the most potent nucleotide analogues were synthesized and showed inhibitory activity against SARS-CoV-2 replication in cell culture.

A small library of novel thiazolidinone-based sulfonamide derivatives was designed, synthesized and evaluated for their ability to target human carbonic anhydrase (hCA) isoforms IX and XII, which are overexpressed in malignant cells and play a key role in metastasis and therapeutic response of cancer cells. A molecular hybridization approach was employed to design the molecules by combining different moieties identified as having antitumor activity. The thiazolidinone core was functionalized with benzenesulfonamide as a zinc-binding group and different isatin derivatives to enhance the chemical profile and optimize the hydrophilic/lipophilic balance. Biological evaluation against hCA I, II, IX and XII isoforms showed promising inhibitory activities, and some compounds exhibited selectivity and high inhibitory activity against hCA IX and hCA XII while not affecting off-target hCA I and hCA II. In particular, compound 3h demonstrated high selectivity with Ki values of 57.8 nM for hCA IX and 44.3 nM for hCA XII.

A small library of 1-(4-nitrophenyl)-3-arylprop-2-en-1-one derivatives was synthesized to identify new human monoamine oxidase B selective inhibitors. Their inhibitory activity toward MAO-A and MAO-B isoforms was evaluated to determine their potency and selectivity. All newly synthesized compounds were nanomolar inhibitors of the B isoform with IC50 concentrations ranging from 120 to 2.2 nM. Conversely, their activity toward the A isozyme was only observed at micromolar concentrations. Our results bear out the hypothesis that the 1,3-diarylpropenone scaffold could represent a valuable starting point for designing efficient and selective MAO-B inhibitors.

Chemotherapeutic agents have remained the first-line treatment option for advanced-stage cancers when surgery or radiation therapy is not viable. Human carbonic anhydrase (hCA) isoforms IX and XII have been validated as anticancer targets. In particular, hCA IX is overexpressed in several solid tumor cells. As a result, selective isoform inhibitors with high potency and low toxicity are sought after. Pursuing our investigation on new scaffolds as hCA-selective inhibitors, a new series of isatin thiazolidinone hybrids has been designed and synthesized. Their biological activity and selectivity toward hCA I, hCA II, hCA IX, and hCA XII were investigated. The results revealed an inhibitory activity in the nanomolar range on carbonic anhydrases IX and XII, and the nature of substitution in positions 3 and 5 of thiazolidinone appears to be crucial for the compounds' selectivity. Docking experiments have been applied to predict the binding mode of these new, promising derivatives.

Human Carbonic Anhydrases (hCA) are enzymes that contribute to cancer’s development and progression. Isoforms IX and XII have been identified as potential anticancer targets, and, more specifically, hCA IX is overexpressed in hypoxic tumor cells, where it plays an important role in reprogramming the metabolism. With the aim to find new inhibitors towards IX and XII isoforms, the hybridization of the privileged scaffolds isatin, dihydrothiazole, and benzenesulfonamide was investigated in order to explore how it may affect the activity and selectivity of the hCA isoforms. In this respect, a series of isatin thiazolidinone hybrids have been designed and synthesized and their biological activity and selectivity on hCA I, hCA II, hCA IX, and hCA XII explored. The new compounds exhibited promising inhibitory activity results on isoforms IX and XII in the nanomolar range, which has highlighted the importance of substituents in the isatin ring and in position 3 and 5 of thiazolidinone. In particular, compound 5g was the most active toward hCA IX, while 5f was the most potent inhibitor of hCA XII within the series. When both potency and selectivity were considered, compound 5f appeared as one of the most promising. Additionally, our investigations were supported by molecular docking experiments, which have highlighted the putative binding poses of the most promising compound.

The Stimulator of Interferon Genes (STING) is involved in cytosolic DNA sensing and type I Interferons (IFN-I) induction. Aiming to identify new STING agonists with antiviral activity and given the known biological activity of benzothiazole and benzimidazole derivatives, a series of benzofuran derivatives were tested for their ability to act as STING agonists, induce IFN-I and inhibit viral replication. Compounds were firstly evaluated in a gene reporter assay measuring luciferase activity driven by the human IFN-β promoter in cells expressing exogenous STING (HEK293T). Seven of them were able to induce IFN-β transcription while no induction of the IFN promoter was observed in the presence of a mutated and inactive STING, showing specific protein-ligand interaction. Docking studies were performed to predict their putative binding mode. The best hit compounds were then tested on human coronavirus 229E replication in BEAS-2B and MRC-5 cells and three derivatives showed EC50 values in the μM range. Such compounds were also tested on SARS-CoV-2 replication in BEAS-2B cells and in Calu-3 showing they can inhibit SARS-CoV-2 replication at nanomolar concentrations. To further confirm their IFN-dependent antiviral activity, compounds were tested to verify their effect on phospho-IRF3 nuclear localization, that was found to be induced by benzofuran derivatives, and SARS-CoV-2 replication in Vero E6 cells, lacking IFN production, founding them to be inactive. In conclusion, we identified benzofurans as STING-dependent immunostimulatory compounds and host-targeting inhibitors of coronaviruses representing a novel chemical scaffold for the development of broad-spectrum antivirals.

Ebola virus (EBOV) is a highly infectious and lethal pathogen responsible for sporadic self-limiting clusters of Ebola virus disease (EVD) in Central Africa capable of reaching epidemic status. 100% protection from lethal EBOV-Zaire in Balb/c mice was achieved by rintatolimod (Ampligen) at the well tolerated human clinical dose of 6 mg/kg. The data indicate that the mechanism of action is rintatolimod's dual ability to act as both a competitive decoy for the IID domain of VP35 blocking viral dsRNA sequestration and as a pathogen-associated molecular pattern (PAMP) restricted agonist for direct TLR3 activation but lacking RIG-1-like cytosolic helicase agonist properties. These data show promise for rintatolimod as a prophylactic therapy against human Ebola outbreaks.

Tumour associated carbonic anhydrases (CAs) IX and XII have been recognised as potential targets for the treatment of hypoxic tumours. Therefore, considering the high pharmacological potential of the chromene scaffold as selective ligand of the IX and XII isoforms, two libraries of compounds, namely 2H-chromene and 7H-furo-chromene derivatives, with diverse substitution patterns were designed and synthesised. The structure of the newly synthesised compounds was characterised and their inhibitory potency and selectivity towards human CA off target isoforms I, II and cancer-associated CA isoforms IX and XII were evaluated. Most of the compounds inhibit CA isoforms IX and XII with no activity against the I and II isozymes. Thus, while the potency was influenced by the substitution pattern along the chromene scaffold, the selectivity was conserved along the series, confirming the high potential of both 2H-chromene and 7H-furo-chromene scaffolds for the design of isozyme selective inhibitors.

The phytochemical investigation of the methanol extract of the seeds of Magydaris pastinacea afforded two undescribed benzofuran glycosides, furomagydarins A-B (1, 2), together with three known coumarins. The structures of the new isolates were elucidated after extensive 1D and 2D NMR experiments as well as HR MS. Compound 1 was able to inhibit the COX-2 expression in RAW264.7 macrophages exposed to lipopolysaccharide, a pro-inflammatory stimulus. RT-qPCR and luciferase reporter assays suggested that compound 1 reduces COX-2 expression at the transcriptional level. Further studies highlighted the capability of compound 1 to suppress the LPS-induced p38MAPK, JNK, and C/EBPβ phosphorylation, leading to COX-2 down-regulation in RAW264.7 macrophages.

In the effort to identify and develop new HIV-1 inhibitors endowed with innovative mechanisms, we focused our attention on the possibility to target more than one viral encoded enzymatic function with a single molecule. In this respect, we have previously identified by virtual screening a new indolinone-based scaffold for dual allosteric inhibitors targeting both reverse transcriptase-associated functions: polymerase and RNase H. Pursuing with the structural optimization of these dual inhibitors, we synthesized a series of 35 new 3-[2-(4-aryl-1,3-thiazol-2-ylidene)hydrazin-1-ylidene]1-indol-2-one and 3-[3-methyl-4-arylthiazol-2-ylidene)hydrazine-1-ylidene)indolin-2-one derivatives, which maintain their dual inhibitory activity in the low micromolar range. Interestingly, compounds 1a, 3a, 10a, and 9b are able to block HIV-1 replication with EC 50 < 20 µM. Mechanism of action studies showed that such compounds could block HIV-1 integrase. In particular, compound 10a is the most promising for further multitarget compound development.

Ebola virus (EBOV) is one of the deadliest infective agents whose lethality is linked to the ability to efficiently bypass the host's innate antiviral response. EBOV multifunctional protein VP35 plays a major role in viral replication both as polymerase cofactor and interferon (IFN) antagonist. By hiding the non-self 5'-ppp dsRNA from the cellular receptor RIG-I, VP35 prevents its activation and inhibits IFN-β production. Blocking VP35-dsRNA interaction and IFN-β suppression is a validated drug target. We screened a library of natural extracts and found that cynarin inhibits dsRNA-VP35 binding with an IC50 value of 8.5 μM. It reverts the EBOV VP35 inhibition of IFN-β production, while it does not induce IFN production by itself. Docking experiments suggest that the molecule can bind on the end-capping pocket of VP35 C-terminal Interferon Inhibitory domain (IID), and differential scanning fluorimetry confirmed that cynarin interacts with VP35-IID with a KD of 12 μM. Cynarin was further tested in an EBOV minigenome assay but did not inhibit VP35 polymerase cofactor activity. When evaluated during challenge of IFN-susceptible A549 cells with EBOV isolate derived from the 2014 West African outbreak, cynarin was able to inhibit viral replication with an EC50 value of 9.1 μM, showing no significant cytotoxicity. Our findings show that cynarin blocks EBOV replication by acting directly on VP35 and subverting its IFN antagonism function but not cofactor function, and as such identify the first EBOV inhibitor with this mode of action.

The field of medicinal chemistry has become increasingly dynamic and medicinal chemists face the challenge of rapidly evolving new technologies.In the last decade, medicinal chemistry methodologies have been largely replaced from an individual scheme to an interdisciplinary approach.Furthermore, the shift from traditional to Omics-based applications is needed to develop computational, chemo, and bioinformatic tools that could help medicinal chemists to analyse, link, and compare the research results.Hence, drug research has necessarily oriented drug discovery toward more rational strategies.In silico Virtual Screening (VS) is one of the most promising approaches to accelerate the drug development process.Efficient analysis of key compounds and target properties is crucial for carrying out a virtual screening process.At the same time, it can reduce the attrition rates in drug development.Of course, the main purpose of VS is to identify novel chemical scaffolds as hits for further optimization using medicinal chemistry approaches.An overview of the most employed methods for VS, challenges, and new directions will be discussed.

Quorum sensing (QS) plays an essential role in the production of virulence factors, in biofilm formation and antimicrobial resistance. Consequently, inhibiting QS is being considered a promising target for antipathogenic/anti-virulence therapies. This study aims to screen 2-nitrovinylfuran derivatives structurally related to Furvina (a broad-spectrum antibiotic already used for therapeutic purposes) for their effects on QS and in biofilm prevention/control. Furvina and four 2-nitrovinylfuran derivatives (compounds 1–4) were tested to assess the ability to interfere with QS of Staphylococcus aureus using bioreporter strains (S. aureus ALC1742 and ALC1743). The activity of Furvina and the most promising quorum-sensing inhibitor (QSI) was evaluated in biofilm prevention and in biofilm control (combined with fusidic acid). The biofilms were further characterized in terms of biofilm mass, viability and membrane integrity. Compound 2 caused the most significant QS inhibition with reductions between 60% and 80%. Molecular docking simulations indicate that this compound interacts preferentially with the protein hydrophobic cleft in the LytTR domain of AgrA pocket. Metabolic inactivations of 40% for S. aureus ALC1742 and 20% for S. aureus ALC1743 were reached. A 24 h-old biofilm formed in the presence of the QSI increased the metabolic inactivation by fusidic acid to 80%, for both strains. The overall results highlight the effects of compound 2 as well as the potential of combining QSI with in-use antibiotics for the management of skin and soft tissues infections.

Current therapeutic protocols for the treatment of HIV infection consist of the combination of diverse anti-retroviral drugs in order to reduce the selection of resistant mutants and to allow for the use of lower doses of each single agent to reduce toxicity. However, avoiding drugs interactions and patient compliance are issues not fully accomplished so far. Pursuing on our investigation on potential anti HIV multi-target agents we have designed and synthesized a small library of biphenylhydrazo 4-arylthiazoles derivatives and evaluated to investigate the ability of the new derivatives to simultaneously inhibit both associated functions of HIV reverse transcriptase. All compounds were active towards the two functions, although at different concentrations. The substitution pattern on the biphenyl moiety appears relevant to determine the activity. In particular, compound 2-{3-[(2-{4-[4-(hydroxynitroso)phenyl]-1,3-thiazol-2-yl} hydrazin-1-ylidene) methyl]-4-methoxyphenyl} benzamide bromide (EMAC2063) was the most potent towards RNaseH (IC50 = 4.5 mM)- and RDDP (IC50 = 8.0 mM) HIV RT-associated functions.

Abstract Bioassay-guided fractionation of the ethyl acetate extract from Teucrium flavum subsp. glaucum, endowed with inhibitory activity towards the HIV-1 reverse transcriptase–associated RNase H function, led to the isolation of salvigenin (1), cirsimaritin (2) and cirsiliol (3) along with the neo-clerodanes teuflavin (4) and teuflavoside (5). Acid hydrolysis of the inactive teuflavoside provided three undescribed neo-clerodanes, flavuglaucins A-C (7-9) and one known neo-clerodane (10). Among all neo-clerodanes, flavuglaucin B showed the highest inhibitory activity towards RNase H function with a IC50 value of 9.1 μM. Molecular modelling and site-directed mutagenesis analysis suggested that flavuglaucin B binds into an allosteric pocket close to RNase H catalytic site. This is the first report of clerodane diterpenoids endowed with anti-reverse transcriptase activity. Neo-clerodanes represent a valid scaffold for the development of a new class of HIV-1 RNase H inhibitors.